【作者】 鲍卫超；

【导师】 刘群；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 近10多年来，鸡球虫病的免疫预防研究越来越受到人们的重视。寻找免疫原性好的保护性抗原，是完成基因工程苗和核酸疫苗研究的前提。微管是球虫的一种重要的细胞器，在球虫入侵宿主细胞的过程中起着重要作用，本论文以E.acervulina的微管蛋白为研究对象，进行了如下试验： 1．E.acervulina α-微管蛋白基因的克隆 以E.acervulina BJ株孢子化卵囊总RNA为模板进行RT-PCR，按照Genebank上发表的α-微管蛋白基因的序列(Yamage M，1995)设计引物，上、下游引物分别加上BamH Ⅰ和EcoR Ⅰ的酶切位点，扩增的PCR产物与pGEM-T载体连接后转化至JM109感受态细胞，蓝白斑筛选，将PCR和酶切鉴定正确的阳性质粒进行测序。结果表明，扩增产物大小为1955bp，与Yamage M(1995)在Genebank上登录的α-微管蛋白基因的同源性为97.4％。此基因现已作为E.acervulinaBJ株的α-微管蛋白基因登录到Genebank上，登录号是AY488134。 2．α-微管蛋白基因的表达及鉴定 重新设计表达引物，以阳性质粒为模板，克隆α-微管蛋白基因的阅读框，与pGEX-6P-1载体连接后转化至E.coli BL21(DE3)表达菌，用1.0mMIPTG诱导，SDS-PAGE电泳后发现在诱导后2h蛋白表达量即达到较高的水平，5h达到高峰，表达蛋白的分子量为69kDa，波层扫描分析表达蛋白占菌体蛋白的33％。用GST抗体进行Western-blot检测，成功检测到特异性条带。 3．α-微管蛋白真核表达载体(p-α-tubulin)的构建 将微管蛋白基因的ORF片段插入到真核表达载体pcDNA3.1中，构建成核酸疫苗pcDNA3.1-α-tubulin(p-α-tubulin)，经酶切和PCR鉴定为正确插入。 4．α-微管蛋白亚单位疫苗和核酸疫苗的免疫保护效果研究 核酸疫苗和亚单位疫苗分别设高(100μg)、中(50μg)、低(25μg)三个剂量组，同时设地克珠利药物组、活卵囊组、红对照组和空白对照组，在鸡只7日龄和14日龄分别进行免疫。一周后用12×10~4个E.acervulina BJ株孢子化卵囊进行攻虫，结果表明α-微管蛋白核酸疫苗中剂量(50μg)免疫时，则可使E.acervulina攻虫鸡的卵囊产量下降34.38％，肠道病变记分降低33.33％，同时鸡只增重效果明显，相对增重率为82.84％，ACI值为164，各指标优于核酸疫苗高剂量和低剂量免疫组。而亚单位疫苗各剂量组对E.acervulina攻虫鸡的效果低于核酸疫苗组。更多还原

【Abstract】 The research to immunizition of the Coccidian was given more and more attention in recent 10 years. Founding the antigens that have better immunity is the precondition to give research on theirs recombinant vaccine and DNA vaccine. Tubulin is one of the important organelles of coccidia, which plays an important role during invading into the cells of host. We focus on the research of a -tubulin and did the tests as follows:1.Clone of the E.acervulina a -tubulin gene The total RNA of the sporulated oocysts of E.acervulina BJ strain was extracted and used as the template for RT-PCR. The primers were designed according to the published a -tubulin sequence on Genebank (Yamage M,1995). The BamHI and EcoR I enzyme cleavage sites were added respectively on the upper and lower primer. The amplified PCR product was linked with the pGEM-T vector, and then transformed into the JM109 competent cell, screened through the blue and white spot, identified by PCR and cleaved with endonucleases, and then the positive clone was sequenced. The results indicated that the amplified gene is 1955bp, the homology is 97.4% between the cloned gene and the published gene on Genebank. Now the gene has been published into Genebank as the alpha-tubulin of E.acervulina BJ strain, the published number is AY488134.2.Expression and identification of the a -tubulin gene The expressed primers were designed anew, The ORF sequence of a -tubulin gene was cloned with the positive plasmid as template. Then the ORF gene was linked with pGEX-6P-l vector and transformed into E.coli BL21(DE3), 1.0mM IPTG was used to induce the expression. The SDS-PAGE result indicated that the protein with the molecular weight 69kDa could be expressed to a high level after being induced 2h, and the level arrived to peak 5h later. The expressed protein accounted for 33% in the protein of bacteria. The specific band was identified with GST antibody through western-blot analysis.3.Construction of the eukaryotic vector (p- a -tubulin) of a -tubulin The DNA vaccine p-a -tubulin was constructed by inserting ORF sequence of a-tubulin into eukaryotic vector pcDNA3.1. The plasmid was identified to be positive plasmid by Enzyme cleavage and PCR.4.Research on the protective effect of a-tubulin DNA vaccine and genetic engineering subunit vaccine Both the a-tubulin DNA vaccine and genetic engineering subunit vaccine were set up three doses groups which were high dose(100 u g), middle dose(50 ug) and low dose(25 u g). At the same time, the Diclazuril group, ooyst group, challenged group and unchallenged group were set up, too. The chicken were immunized respectively on the 7th and 14th day of age and challenged with 12×104sporulated oocysts of E.acervulina a week later. Results indicated that the DNA vaccine that used in middle dose reduced the oocysts output 34.38% to the chicken challenged with E.acervulina, the lesion scores of intestine decreased 33.03%, at the same time, the weight gains were obvious, relative weight gain ratio was 82.84%. ACI was 164. All of the indexes were better than the high dose and low dose of DNA vaccine gourps. However, the protective effects of all doses of genetic engineering subunit vaccine groups were lower than the protective effects of DNA vaccine groups.更多还原